VPR receptor protein

ABSTRACT

A human receptor protein which binds to the human immunodeficiency virus (HIV) viral protein R (vpr) is disclosed. Pharmaceutical compositions that comprise the receptor protein, compositions useful to produce the receptor protein and methods of making and using the receptor protein are disclosed.

This application is a National Stage application of PCT/US94/14532,filed under 35 U.S.C. §371 on Dec. 15, 1994, which is acontinuation-in-part application of Ser. No. 08/167,519, filed Dec. 15,1993, now abandoned.

FIELD OF THE INVENTION

The present invention relates to a human receptor protein which binds tothe human immunodeficiency virus (HIV) viral protein R (vpr), topharmaceutical compositions that comprise the receptor protein, tocompositions useful to produce the receptor protein and to methods ofmaking and using the receptor protein. The present invention is relatedto U.S. application Ser. No. 08/167,519 filed Dec. 15, 1993, which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

Since the demonstration in 1987 that the small open reading frame withinHIV-1 designated R encodes a 15 kd protein (Wong-Staal, F., et al.,(1987) AIDS Res. Hum. Retroviruses 3:33-39), relatively little regardingthe function of the viral protein R (vpr) has been reported. The vpropen reading frame is conserved within all genomes of HIV-1 and HIV-2and within most, if not all, simian immunodeficiency virus (SIV)genomes. VPR is immunogenic in vivo in that a large subset of HIVindividuals makes antibodies that can react with a bacterially producedvpr peptide (Wong-Staal, F., et al. , (1987) AIDS Res. Hum. Retroviruses3:33-39).

The progression from HIV infection to AIDS is in large part determinedby the effects of HIV on the cells that it infects, including CD4⁺ Tlymphocytes and macrophages. On the other hand, cell activation,differentiation and proliferation are in turn thought to regulate HIVinfection and replication in T cells and macrophages. Gallo, R. C. etal. (1984) Science 224:500; Levy, J. A. et al., (1984) Science 225:840;Zack, J. A. et al. (1988) Science 240:1026; Griffin, G. E. et al.,(1988) Nature 339:70; Valentin, A. et al. (1991) J. AIDS 4:751; Rich, E.A. et al., (1992) J. Clin. Invest. 89:176; and Schuitemaker, H. et al.(1992) J. Virol. 66:1354. Cell division per se may not be required sinceHIV and other lentiviruses can proliferate in nonproliferating,terminally differentiated macrophages and growth-arrested T lymphocytes.Rose, R. M. et al. (1986) Am. Rev. Respir. Dis. 143:850; Salahuddin, S.Z. et al. (1986) Blood 68:281; and Li, G. et al. (1993) J. Virol.67:3969. The ability of lentiviruses, including HIV, to replicate innonproliferating cells, particularly in macrophages, is believed to beunique among retroviruses and it may be significant that severallentiviruses contain a vpr-like gene. Myers, G. et al. (1992) AIDS Res.Hum. Retrovir. 8:373. HIV infection of myeloid cell lines can result ina more differentiated phenotype and increase the expression of factorssuch as NF-KB which are necessary for HIV replication. Roulston, A. etal. (1992) J. Exp. Med. 175:751; and Chantal Petit, A. J. et al. (1987)J. Clin. Invest. 79:1883.

The most evidence for the function of the vpr protein comes from severalstudies reporting the activities of HIV strains that have mutations inthe vpr gene. It has been reported that mutations in the vpr generesults in a decrease in the replication and cytopathogenicity of HIV-1,HIV-2, and SIV in primary CD4⁺ T lymphocytes and transformed T celllines (Ogawa, K., et al., (1989) J. Virol. 63:4110-4114; Shibata, R., etal. (1990a). J. Med. Primatol. 19:217-225; Shibata, R., et al. (1990b)J. Virol. 64:742-747 and Westervelt, P. et al. (1992) J. Virol.66:3925), although others have reported mutated vpr gene had no effecton replication (Dedera, D., et al. (1989) Virol. 63:3205-3208).Interestingly HIV-2 mutated for vpr has been reported unable to infectprimary monocyte/macrophages (Hattori, N., et al. (1990) Proc. Nati.Acad. Sci. U.S.A. 87:8080-8084). Transactivation of the HIV longterminal repeat and heterologous promoters by HIV is increased about3-fold in wild-type versus vpr-negative HIV-1, though the mechanismthrough which vpr may transactivate transcription is unknown and may beindirect (Cohen, E. A., et al., (1990b) J. Acquir. Immune Defic. Syndr.3:11-18). The relationship between the effects of vpr on promoteractivity and viral infectivity is not clear. Vpr protein is incorporatedinto the viral particle, and this finding has led to the propositionthat vpr functions early in infection, following virus penetration anduncoating, and that vpr may interact with cellular regulatory mechanismsimportant in the establishment of infection (Cohen, E. A., et al. 1990aJ. Virol. 64:3097-3099; Yu, X. F., et al. (1990) J. Virol.64:5688-5693.; and, Yuan, X., et al., (1990) AIDS Res. Hum. Retroviruses6:1265-1271).

The vpr gene of HIV-1 has been shown to induce cellular growthinhibition and differentiation in tumor lines of intermediatedifferentiation in vitro. Levy, D. N. et al. (1993) Cell 72:541. Sincevpr protein originates within viral particles, vpr may play a role inestablishing productive infection.

There is a need to understand the activity of vpr and its role in HIVinfection at the molecular and cellular level. There is a need toidentify the cellular proteins that bind to vpr. There is a need toidentify molecules that inhibit vpr activity. There is a need foranti-HIV therapeutics and protective agents.

SUMMARY OF THE INVENTION

The present invention relates to essentially pure human protein which isthe receptor of HIV vpr, that is it binds to vpr. The protein whenunbound to vpr occurs in the cytoplasm of human cells and is transportedfrom the cytoplasm to the nucleus when bound to vpr. The protein has anapparent molecular weight of between 40-43 kD. Using 12% matrices inSDS-PAGE, the observed molecular weight of the protein is 41 kD. Theprotein binds to human glucocorticoid receptor protein. The presentinvention relates to compositions having about 95% pure vpr receptorprotein.

The present invention relates to fragments of the vpr receptor whichbind to vpr.

The present invention relates to a method of identifying compounds whichinhibit vpr protein binding to the vpr receptor protein that comprisesthe steps of first contacting vpr protein and the vpr receptor proteinor a fragment thereof which binds to vpr in the presence of a testcompound, then determining the level of binding band then comparing thatlevel to the level of binding that occurs when vpr protein and the vprreceptor protein are contacted in the absence of a test compound.

The present invention relates to a kit for identifying compounds whichinhibit vpr protein binding to the vpr receptor protein which comprisesa first container comprising vpr protein and a second containercomprising the vpr receptor protein or a fragment thereof which binds tovpr.

The present invention relates to antibodies that specifically bind tothe vpr receptor protein.

The present invention relates to pharmaceutical compositions thatcomprise the vpr receptor protein or a fragment thereof which binds tovpr, and a pharmaceutically acceptable carrier.

The present invention relates to a method of treating an individualexposed to HIV by administering the vpr receptor protein.

DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present invention arises out of the discovery that HIV regulatoryprotein vpr (referred to herein as "vpr" or "vpr protein") binds toprotein found in the cytoplasm of human cells. The human protein,referred to herein as "vpr receptor protein", has an apparent molecularweight of between 40-43 kD. The vpr receptor protein has been observedto have a molecular weight of about 41 kD as determined using 12%SDS-PAGE. The vpr receptor protein has the ability to bind to vpr. Thevpr receptor protein has the ability to bind to the human glucocorticoidreceptor. The vpr receptor protein is soluble in Triton. It has beendiscovered that when vpr binds to the vpr receptor protein in cells, thevpr receptor protein which is normally found in the cytoplasm of humancells and the vpr protein bound to it are transported from the cytoplasmto the nucleus. The vpr receptor protein is colocalized with the T-celland B-cell transcription factor NFkB.

As described in U.S. patent application Ser. No. 08/019,601 filed Feb.19, 1993 entitled VPR Function and Activity and the U.S. PatentApplication filed herewith which is entitled VPR Function and Activityand which is a continuation in part of U.S. patent application Ser. No.08/167,519, both of which are incorporated herein by reference, vpr hasseveral activities which are involved in HIV infection. In particular,vpr is believed to enhance retroviral infection by causing changes incells that make them better hosts for HIV replication.

The discovery of the vpr receptor protein in human cells and itstransport from cytoplasm to the nucleus when bound to vpr indicate thatthe binding of vpr to the human receptor protein is involved in HIVreplication and thus pathogenesis. Accordingly, the inhibition of suchinteraction effectively inactivates vpr and prevents it from convertingcells to better HIV replication hosts.

The present invention relates to essentially pure human vpr receptorprotein. The vpr receptor protein can be isolated from human cells bypassing a human cell preparation through an immobilized vpr column underconditions which allow vpr/vpr receptor binding and then changing theconditions to those which do not favor such binding. The released vprreceptor can be collected in essentially pure form. Further purificationmay be achieved using routine chromatography means.

The following procedure may be used to purify vpr receptor proteins.Cell extracts from primary T cells and monocytes as well as peripheralblood cells and macrophages are obtained by methods known to thoseskilled in the art. Cell extracts are separated by affinitychromatography. Briefly, eukaryotically-produced vpr is immobilized to asolid support matrix via one or more covalent bonds. Solid supportmatrices include agarose, polyacrylamide-agarose, controlled-pore glassand other such materials known to those skilled in the art. One skilledin the art will readily appreciate the standard techniques involved incoupling vpr to the matrix as well as techniques involved in activationof the matrix. A spacer molecule may be employed to distance vpr fromthe matrix backbone in order to allow vpr to more freely bind proteinsin the cell extract. One skilled in the art will readily appreciate thevariety of spacer molecules with which to use.

The cell extract is layered onto the vpr affinity column by standardmethods known to those skilled in the art. Appropriate buffers, washingconditions and elution conditions, which are known to those skilled inthe art, are chosen. The resulting eluate may be further purified tohomogeneity by techniques such high performance liquid chromatography(HPLC) or other such methods as known to those skilled in the art.

The vpr receptor protein has been purified to approximately 95% purityby a vpr-affinity column using this technique of purification. Theprotein has a molecular weight of about 40-43 kDa when separated byreducing SDS-PAGE. Using SDS-PAGE with a 12% electrophoresis matrix, thevpr receptor was observed to migrate with a molecular weight of 41 kD.The protein has been detected in rhabdomyosarcoma cell lines TE 671 andRD; osteosarcoma cell lines D17 and HOS; glioblastoma cell lines HTB14,U373 and HBT10; as well as T-cell lines Supt-1 and H9 andmonocyte/macrophage lines U937, THP-1, KG-1 and HL-60 as well as primarycells.

Techniques for the cloning of a protein are widely known to thoseskilled in the art. Briefly, a pure preparation of the 41 kDa cellularprotein that binds vpr is sequenced by standard N-terminal sequencingtechniques known to those skilled in the art. A set of oligonucleotideprobes coding for the deduced amino acid sequence of the N-terminalportion of the 41 kDa protein is designed by techniques known to thoseskilled in the art. This set of probes is used to screen a human CDNAlibrary by techniques known to those skilled in the art. Positiveplaques are selected and sequenced by methods such as dideoxy sequencingfor the entire nucleotide sequence of the 41 kDa protein.

Alternatively, a pure preparation of the 41 kDa protein may be injectedinto a mammal, such as a rabbit, resulting in the production of apolyclonal antiserum. Such immunization procedures are well known tothose skilled in the art. In addition, plasma cells (antibody-producingB cells) may be isolated from the injected mammal and fused with myelomacells to produce hybridomas which produce monoclonal antibodies. Suchmethods are well known to those skilled in the art. The polyclonalantiserum may be used to screen a human cDNA expression library whereincells expressing the 41 kDa protein may be identified with theantiserum. Positive clones are selected and the DNA isolated andsequenced by methods known to those skilled in the art.

Once the complete nucleotide sequence of the 41 kDa cellular protein isknown, the sequence, or any portion thereof, can be incorporated into aplasmid vector or any other vector capable of expressing the 41 kDaprotein. In addition, mammalian cells as well as bacterial cells may betransformed with the plasmid construct containing the sequence, orderivatives thereof, encoding the 41 kDa protein. Said transformed cellsmay produce the 41 kDa protein intracellularly or extracellularly. Inaddition, oligonucleotides corresponding to the portions of the sense orantisense of the 41 kDa protein may also be produced. Theseoligonucleotides may comprises between 10 and 5000 nucleotides,preferably between 10 and 500 nucleotides, most preferably between 10and 100 nucleotides.

The present invention also relates to: a nucleic acid molecule thatcomprises a nucleotide sequence that encodes vpr receptor protein or afragment thereof; an expression vector that comprises a nucleotidesequence that encodes vpr receptor protein or a fragment thereof; a hostcell which comprises the expression vector; and a method of producing avpr receptor protein or a fragment thereof comprising the step ofculturing the host cell.

Vpr receptor protein may be produced by routine means using readilyavailable starting materials as described above. Provision of a suitableDNA sequence encoding the desired protein permits the production of theprotein using recombinant techniques now known in the art. The DNAsequence may also be obtained from other sources of HIV DNA or can beprepared chemically using a synthesized nucleotide sequence. When thecoding DNA is prepared synthetically, advantage can be taken of knowncodon preferences of the intended host where the DNA is to be expressed.

One having ordinary skill in the art can, using well known techniques,obtain a DNA molecule encoding the vpr receptor protein and insert thatDNA molecule into a commercially available expression vector for use inwell known expression systems. For example, the commercially availableplasmid pSE420 (Invitrogen, San Diego, Calif.) may be used forproduction in E. coli. The commercially available plasmid pYES2(Invitrogen, San Diego, Calif.) may be used for production in S.cerevisiae strains of yeast. The commercially available MaxBac™(Invitrogen, San Diego, Calif.) complete baculovirus expression systemmay be used for production in insect cells. The commercially availableplasmid pcDNA I (Invitrogen, San Diego, Calif.) may be used forproduction in may be used for production in mammalian cells such asChinese Hamster Ovary cells. One having ordinary skill in the art canuse these commercial expression vectors systems or others to produce vprreceptor protein using routine techniques and readily available startingmaterials.

One having ordinary skill in the art may use other commerciallyavailable expression vectors and systems or produce vectors using wellknown methods and readily available starting materials. Expressionsystems containing the requisite control sequences, such as promotersand polyadenylation signals, and preferably enhancers, are readilyavailable and known in the art for a variety of hosts. See e.g.,Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. ColdSpring Harbor Press (1989). Thus, the desired proteins can be preparedin both prokaryotic and eukaryotic systems, resulting in a spectrum ofprocessed forms of the protein.

The most commonly used prokaryotic system remains E. coli, althoughother systems such as B. subtilis and Pseudomonas are also useful.Suitable control sequences for prokaryotic systems include bothconstitutive and inducible promoters including the lac promoter, the trppromoter, hybrid promoters such as tac promoter, the lambda phage P1promoter. In general, foreign proteins may be produced in these hostseither as fusion or mature proteins. When the desired sequences areproduced as mature proteins, the sequence produced may be preceded by amethionine which is not necessarily efficiently removed. Accordingly,the peptides and proteins claimed herein may be preceded by anN-terminal Met when produced in bacteria. Moreover, constructs may bemade wherein the coding sequence for the peptide is preceded by anoperable signal peptide which results in the secretion of the protein.When produced in prokaryotic hosts in this matter, the signal sequenceis removed upon secretion.

A wide variety of eukaryotic hosts are also now available for productionof recombinant foreign proteins. As in bacteria, eukaryotic hosts may betransformed with expression systems which produce the desired proteindirectly, but more commonly signal sequences are provided to effect thesecretion of the protein. Eukaryotic systems have the additionaladvantage that they are able to process introns which may occur in thegenomic sequences encoding proteins of higher organisms. Eukaryoticsystems also provide a variety of processing mechanisms which result in,for example, glycosylation, carboxy-terminal amidation, oxidation orderivatization of certain amino acid residues, conformational control,and so forth.

Commonly used eukaryotic systems include, but is not limited to, yeast,fungal cells, insect cells, mammalian cells, avian cells, and cells ofhigher plants. Suitable promoters are available which are compatible andoperable for use in each of these host types as well as are terminationsequences and enhancers, as e.g. the baculovirus polyhedron promoter. Asabove, promoters can be either constitutive or inducible. For example,in mammalian systems, the mouse metallothionene promoter can be inducedby the addition of heavy metal ions.

The particulars for the construction of expression systems suitable fordesired hosts are known to those in the art. For recombinant productionof the protein, the DNA encoding it is suitably ligated into theexpression vector of choice and then used to transform the compatiblehost which is then cultured and maintained under conditions whereinexpression of the foreign gene takes place. The protein of the presentinvention thus produced is recovered from the culture, either by lysingthe cells or from the culture medium as appropriate and known to thosein the art.

One having ordinary skill in the art can, using well known techniques,isolate the vpr receptor protein or fragments thereof produced usingsuch expression systems.

In addition to isolating vpr receptor protein from natural sources andproducing vpr receptor protein or fragments thereof by recombinanttechniques, automated amino acid synthesizers may also be employed toproduce vpr receptor protein or fragments thereof. It should be furthernoted that if the proteins herein are made synthetically, substitutionby amino acids which are not encoded by the gene may also be made.Alternative residues include, for example, the ω amino acids of theformula H₂ N(CH₂)_(n) COOH wherein n is 2-6. These are neutral, nonpolaramino acids, as are sarcosine (Sar), t-butylalanine (t-BuAla),t-butylglycine (t-BuGly), N-methyl isoleucine (N-MeIle), and norleucine(Nleu). Phenylglycine, for example, can be substituted for Trp, Tyr orPhe, an aromatic neutral amino acid; citrulline (Cit) and methioninesulfoxide (MSO) are polar but neutral, cyclohexyl alanine (Cha) isneutral and nonpolar, cysteic acid (Cya) is acidic, and ornithine (Orn)is basic. The conformation conferring properties of the proline residuesmay be obtained if one or more of these is substituted by hydroxyproline(Hyp).

The pharmaceutical composition comprising vpr receptor protein or afragment thereof and a pharmaceutically acceptable carrier or diluentmay be formulated by one having ordinary skill in the art withcompositions selected depending upon the chosen mode of administration.Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field.

For parenteral administration, the vpr receptor protein or a fragmentthereof can be, for example, formulated as a solution, suspension,emulsion or lyophilized powder in association with a pharmaceuticallyacceptable parenteral vehicle. Examples of such vehicles are water,saline, Ringer's solution, dextrose solution, and 5' human serumalbumin. Liposomes and nonaqueous vehicles such as fixed oils may alsobe used. The vehicle or lyophilized powder may contain additives thatmaintain isotonicity (e.g., sodium chloride, mannitol) and chemicalstability (e.g., buffers and preservatives). The formulation issterilized by commonly used techniques. For example, a parenteralcomposition suitable for administration by injection is prepared bydissolving 1.5% by weight of active ingredient in 0.9% sodium chloridesolution.

The pharmaceutical compositions according to the present invention maybe administered as a single doses or in multiple doses. Thepharmaceutical compositions of the present invention may be administeredeither as individual therapeutic agents or in combination with othertherapeutic agents. The treatments of the present invention may becombined with conventional therapies, which may be administeredsequentially or simultaneously.

The pharmaceutical compositions comprising vpr receptor protein, orfragments or derivatives may be administered by any means that enablesthe active agent to reach the agent's site of. action in the body of amammal. The dosage administered varies depending upon factors such as:pharmacodynamic characteristics; its mode and route of administration;age, health, and weight of the recipient; nature and extent of symptoms;kind of concurrent-treatment; and frequency of treatment. Usually, adaily dosage of vpr receptor protein can be about 1 μg to 100 milligramsper kilogram of body weight. Ordinarily 0.5 to 50, and preferably 1 to10 milligrams per kilogram per day given in divided doses 1 to 6 times aday or in sustained release form is effective to obtain desired results.

Another aspect of the present invention relates to pharmaceuticalcompositions that comprise a nucleic acid molecule that encodes vprreceptor protein or a fragment thereof and a pharmaceutically acceptablecarrier or diluent. According to the present invention, genetic materialthat encodes vpr receptor protein or a fragment thereof is delivered toan individual in an expressible form. The genetic material, DNA or RNA,is taken up by the cells of the individual and expressed. Pharmaceuticalcompositions comprising genetic material that encodes vpr receptorprotein are useful in the same manner as pharmaceutical compositionscomprising vpr receptor protein.

The pharmaceutical compositions according to this aspect of the presentinvention comprise about 0.1 to about 1000 micrograms of DNA. In somepreferred embodiments, the pharmaceutical compositions contain about 1to about 500 micrograms of DNA. In some preferred embodiments, thepharmaceutical compositions contain about 25 to about 250 micrograms ofDNA. Most preferably, the pharmaceutical compositions contain about 100micrograms DNA.

The pharmaceutical compositions according to this aspect of the presentinvention are formulated according to the mode of administration to beused. One having ordinary skill in the art can readily formulate anucleic acid molecule that encodes vpr receptor protein. In cases whereintramuscular injection is the chosen mode of administration, anisotonic formulation is used. Generally, additives for isotonicity caninclude sodium chloride, dextrose, mannitol, sorbitol and lactose.Isotonic solutions such as phosphate buffered saline may be used.Stabilizers include gelatin and albumin.

The present invention relates to a method of treating an individualexposed to HIV by administering pharmaceutical compositions thatcomprise the vpr receptor protein or fragments thereof. Solublefragments which bind to vpr are particularly useful.

Another aspect of the invention relates to methods of identifyingcompounds which inhibit vpr protein binding to vpr receptor protein. Themethods comprise the steps of first contacting, in the presence of atest compound, vpr protein and vpr receptor protein and then determiningthe level of binding. Compounds which interfere with the binding of vprto vpr receptor protein are useful to impede HIV replication; thereforesuch compounds will be useful as anti-HIV therapeutics alone or as partof a multi-faceted anti-HIV drug regimen which includes othertherapeutics.

To practice these aspects of the invention, vpr protein and vpr receptorprotein are contacted in the presence of a test compound. The level ofbinding of the proteins is determined. The resultant level of binding iscompared to the known level of binding that occurs when both proteinsare contacted with each other. In the absence of a compound thatinterferes with the binding, the two proteins will bind. As a control,vpr protein and vpr receptor protein are contacted in the absence of atest compound.

Test compound is provided, preferably in solution. Serial dilutions oftest compounds may be used in a series of assays. Test compound may beadded at concentrations from 0.01 μM to 1M. A preferred range of finalconcentrations of a test compound is from 1.0 μM to 100 μM.

Production of vpr protein is described in the U.S. Patent Applicationcited above which have been incorporated by reference. A preferredconcentration range of the vpr used is about 1 μg/ml to 1 mg/ml. Apreferred concentration of the vpr is about 50 μg/ml.

The vpr receptor protein may be produced by routine means using readilyavailable starting materials following the teachings described herein. Apreferred concentration range of the vpr receptor protein used is about1 μg/ml to 1 mg/ml. A preferred concentration of the vpr receptorprotein is about 50 μg/ml.

The means to detect whether or not vpr and vpr receptor protein arebound or if binding has been inhibited are routine and include enzymeassays and ELISA assays. One having ordinary skill in the art can detectprotein binding using well known methods. One having ordinary skill inthe art can readily appreciate the multitude of ways to practice abinding assay to detect compounds which modulate the binding of vpr tovpr receptor protein. For example, antibodies are useful forimmunoassays which detect or quantitate vpr protein binding to vprreceptor protein. The immunoassay typically comprises incubating vprprotein and vpr receptor protein to allow protein-protein binding in thepresence of a detectably labeled high affinity antibody capable ofselectively binding to either vpr protein or vpr receptor protein, anddetecting the labeled antibody which is bound to the protein. Variousimmunoassay procedures are described in Immunoassays for the 80's, A.Voller et al., Eds., University Park, 1981.

In this aspect of the invention, the antibody or either vpr protein orvpr receptor protein may be added to nitrocellulose, or other solidsupport which is capable of immobilizing proteins. The support may thenbe washed with suitable buffers followed by treatment with thedetectably labeled vpr-specific antibody or the antibody that binds tothe vpr receptor protein. The solid phase support may then be washedwith the buffer a second time to remove unbound antibody. The amount ofbound label on said solid support may then be detected by conventionalmeans.

By "solid phase support" or "carrier" is intended any support capable ofbinding antigen or antibodies. Well-known supports or carriers, includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, agaroses,and magnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport configuration may be spherical, as in a bead, or cylindrical, asin the inside surface of a test tube, or the external surface of a rod.Alternatively, the surface may be flat such as a sheet, test strip, etc.Those skilled in the art will know many other suitable carriers forbinding antibody or antigen, or will be able to ascertain the same byuse of routine experimentation.

The binding activity of a given lot of antibodies may be determinedaccording to well known methods. Those skilled in the art will be ableto determine operative and optimal assay conditions for eachdetermination by employing routine experimentation.

Positive control assays may be performed in which no test compound isadded.

One of the ways in which the antibodies can be detectably labeled is bylinking the same to an enzyme and use in an enzyme immunoassay (EIA), orenzyme-linked immunosorbent assay (ELISA). This enzyme, whensubsequently exposed to its substrate, will react with the substrategenerating a chemical moiety which can be detected, for example, byspectrophotometric, fluorometric or by visual means. Enzymes which canbe used to detectably label the antibody include, but are not limitedto, malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphatedehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, betagalactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase.

By radioactively labeling the antibody, it is possible to detect itthrough the use of a radioimmunoassay (RIA) (see, for example, Work, T.S., et al., Laboratory Techniques and Biochemistry in Molecular Biology,North Holland Publishing Company, N.Y., 1978. The radioactive isotopecan be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography. Isotopes which areparticularly useful for the purpose of the present invention are: ³ H,¹²⁵ I, ¹³¹ I, ³⁵ S, ¹⁴ C, and, preferably, ¹²⁵ I.

It is also possible to label the antibody with a fluorescent compound.When the fluorescent labeled antibody is exposed to light of the properwave length, its presence can then be detected due to fluorescence.Among the most commonly used fluorescent labelling compounds arefluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence-emittingmetals such as ¹⁵² Eu, or others of the lanthanide series. These metalscan be attached to the TNF-specific antibody using such metal chelatinggroups as diethylenetriaminepentaacetic acid (DTPA) orethylenediamine-tetraacetic acid (EDTA).

The antibody also can be detectably labeled by coupling to achemiluminescent compound. The presence of the chemiluminescentlylabeled antibody is then determined by detecting the presence ofluminescence that arises during the course of a chemical reaction.Examples of particularly useful chemiluminescent labeling compounds areluminol, isoluminol, theromaticacridiniumester, imidazole, acridiniumsalt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody.Bioluminescence is a type of chemiluminescence found in biologicalsystems in which a catalytic protein increases the efficiency of thechemiluminescent reaction. The presence of a bioluminescent protein isdetermined by detecting the presence of luminescence. Importantbioluminescent compounds for purposes of labeling are luciferin,luciferase and aequorin. Detection of the vpr-specific antibody or theantibody that binds to the vpr receptor protein may be accomplished by ascintillation counter, for example, if the detectable label is aradioactive gamma emitter, or by a fluorometer, for example, if thelabel is a fluorescent material.

In the case of an enzyme label, the detection can be accomplished bycolorometric methods which employ a substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

As can be readily appreciated, one of the viral proteins may also bedetectable and serve as a reporter molecule instead of or in addition tothe antibody.

The components of the assay may be adapted for utilization in animmunometric assay, also known as a "two-site" or "sandwich" assay. In atypical immunometric assay, a quantity of unlabeled antibody (orfragment of antibody) is bound to a solid support that is insoluble inthe fluid being tested and a quantity of detectably labeled solubleantibody is added to permit detection and/or quantitation of the ternarycomplex formed between solid-phase antibody, antigen, and labeledantibody.

Typical and preferred immunometric assays include "forward" assays inwhich the antibody bound to the solid phase is first contacted with theone of the viral proteins to immobilize it. The second viral protein isadded in the presence of the test compound. After a suitable incubationperiod, the solid support is washed to remove unbound protein. A secondantibody is then added which is specific for the second viral protein.The second antibody is preferably detectable. After a second incubationperiod to permit the labeled antibody to complex with the second viralprotein bound to the solid support through the unlabeled antibody andfirst viral protein, the solid support is washed a second time to removethe unreacted labeled antibody. This type of forward sandwich assay maybe a simple "yes/no" assay to determine whether binding has occurred ormay be made quantitative by comparing, the measure of labeled antibodywith that obtained in a control. Such "two-site" or "sandwich" assaysare described by Wide, Radioimmune Assay Method, Kirkham, Ed., E. & S.Livingstone, Edinburgh, 1970 pp. 199-206).

Other type of "sandwich" assays are the so-called "simultaneous" and"reverse" assays. A simultaneous assay involves a single incubation stepwherein the antibody bound to the solid support and labeled antibody,both viral protein and the test compound are added at the same time.After the incubation is completed, the solid support is washed to removeuncomplexed proteins. The presence of labeled antibody associated withthe solid support is then determined as it would be in a conventional"forward" sandwich assay.

In the "reverse" assay, stepwise addition first of a solution of labeledantibody to the viral proteins followed by the addition of unlabeledantibody bound to a solid support after a suitable incubation period, isutilized. After a second incubation, the solid phase is washed inconventional fashion to free it of the residue of the sample beingtested and the solution of unreacted labeled antibody. The determinationof labeled antibody associated with a solid support is then determinedas in the "simultaneous" and "forward" assays. In one embodiment, acombination of antibodies of the present invention specific for separateepitopes may be used to construct a sensitive three-siteimmunoradiometric assay.

In some preferred embodiments, an anti-vpr antibody is fixed to a solidphase. vpr protein is contacted with the fixed antibody to form acomplex. The complex is contacted with a vpr receptor protein in thepresence of a test compound. Antibodies that. bind to the vpr receptorprotein are then added. The solid phase is washed to removed unboundmaterial. A control assay is performed in an identical manner exceptthat no test compound is used. Detection of the antibodies that bind tothe vpr receptor protein indicates that the vpr and vpr receptorproteins are capable of binding to each other in the presence of thetest compound. Accordingly, failure to detect that antibodies that bindto vpr protein indicates that the test compound inhibits binding of vprand vpr receptor proteins. Quantifying the level of binding in thepresence and absence of test compound allows for the measurement of theextent of modulation that the test compound can cause on vpr binding tovpr receptor protein.

In some preferred embodiments, antibodies that bind to the vpr receptorprotein are fixed to a solid phase. vpr receptor protein is contactedwith the fixed antibody to form a complex. The complex is contacted withvpr protein in the presence of a test compound. Anti-vpr antibodies arethen added. The solid phase is washed to removed unbound material. Acontrol assay is performed in an identical manner except that no testcompound is used. Detection of the antibodies that bind to vpr proteinindicates that the vpr and vpr receptor proteins are capable of bindingto each other in the presence of the test compound. Accordingly, failureto detect that antibodies that bind to vpr protein indicates that thetest compound inhibits binding of vpr and vpr receptor proteins.Quantifying the level of binding in the presence and absence of testcompound allows for the measurement of the extent of modulation that thetest compound can cause on vpr binding to vpr receptor protein.

In the methods of identifying compounds that inhibit vpr protein bindingto vpr receptor protein, fragments of vpr may be used provided thefragment used retains its ability to bind to the vpr receptor protein.Similarly, fragments of vpr receptor protein may be used provided thefragment used retains its ability to bind to vpr protein.

A further aspect of the present invention relates to kits for practicingthe above described method of identifying compounds which inhibit vprprotein binding to vpr receptor protein. Kits according to this aspectof the invention comprises a first container comprising vpr protein, asecond container comprising vpr receptor protein. Additionally, topractice the above defined method, means are required to distinguish vprprotein bound to the vpr receptor protein from unbound vpr protein orunbound vpr receptor protein. In a preferred embodiment of this aspectof the invention, a third container comprising an antibody thatspecifically binds to either the vpr protein or vpr receptor protein isprovided. At least one of the contained components, preferably theantibody, may be conjugated with an agent, such as those describedabove, which allows its presence to be detected. In another preferredembodiment of this aspect of the invention, a fourth container isprovided which contains an antibody that specifically binds to eitherthe vpr protein or vpr receptor protein, but not the protein which isbound by the antibody in the third container. At least one of thecontained components, preferably the antibody, may be conjugated with anagent, such as those described above, which allows its presence to bedetected. In the kits of the invention which are useful to practice themethods of identifying compounds that inhibit vpr protein binding to aprotein, fragments of vpr may be included provided the fragment usedretains its ability to bind to the vpr receptor protein. Similarly,fragments of vpr receptor protein may be included provided the fragmentused retains its ability to bind to vpr protein.

The present invention relates to antibodies that specifically bind tothe human protein that has an apparent molecular weight of between 40-43kD, that occurs in the cytoplasm of human cells, that binds to vpr andthat is transported from the cytoplasm to the nucleus when bound to vpr.Production of such antibodies can be achieved by those having ordinaryskill in the art without undue experimentation using readily availablestarting materials. The antibodies are useful in the assay to identifycompounds that inhibit vpr binding to vpr receptor protein.

EXAMPLES Example 1

Supernatants that contained vpr protein from insect cells infected withrecombinant baculovirus that comprised a nucleotide sequence thatencodes vpr were passed over a column. The column was washed with PBS.Cell lysates from U937 cells lysed in 100 mM NaCl, 50 mM Tris pH 8.0,0.5% triton X-100 were then passed over the vpr loaded column and washedwith PBS. The column was eluted with 100 mM triethanolamine, pH 11.5.The eluate was neutralized with 1M sodium phosphate, pH 6.8. Vpr and vprreceptor protein were coeluted as concluded from ELISA, SDS PAGE, silverstain or western blot.

vpr receptor protein was further purified by adding rabbit anti-vprcoated beads to the eluate. The beads are covalently bound to theantibody and the elution is done with the same triethanolamine solutiondescribed above. The beads are washed with PBS and eluted. The eluate issupplemented with equimolar amounts of gag p24. This solution isincubated at room temperature for 30 minutes. After incubation, beadscoupled to a different anti-vpr antibody as well as antibody V7.8 areadded to the solution. The supernatant is collected and contains greaterthan 90% pure vpr receptor protein which can be further purified bychromatography.

Example 2

Peptides that consist of vpr residues 27-39, 35-48, 41-55, 49-60 and66-68 inhibit vpr/vpr receptor binding. Anti-vpr antibody binds to vprpeptide 41-60 inhibits vpr/vpr receptor binding.

Example 3

The following procedure was used to obtain substantially pure vprreceptor protein. Since the vpr receptor protein is Triton soluble, itcan be purified from Triton soluble portions of cell lysates. The vprreceptor protein binds to vpr. Accordingly, using a column packed withvpr, the receptor protein can be isolated from Triton soluble portionsof cell lysates and eluted as an isolated protein.

Recombinant vpr was produced in insect cells using baculovirusexpression as described in Levy et al. (1994) Proc. Natl. Acad. Sci.U.S.A. 91:10873-10877, which is incorporated herein by reference.Recombinant vpr was purified by adding Triton X-100 at 0.05% v/v finalconcentration to the baculovirus supernatants. The supernatants werethen passed through a rabbit anti-vpr column as described in Levy et al.(1994) Proc. Natl. Acad. Sci. U.S.A. 91:10873-10877 which wasconstructed in Harlow E. and E. Lane Antibodies: A laboratory manualCold Spring Harbor Press, Cold Spring Harbor, N.Y. 1988, which isincorporated herein by reference. After extensive washing with PBSTriton X-100, the columns, the columns were eluted as follows. Threebeds volumes of a pre-elution buffer at 10 mM Sodium Phosphate bufferplus Triton X-100 (0.05%), pH 8.0, were passed through the column,followed by the elution buffer consisting of 10 mM Triethanolamine plus0.05% Triton X-100, pH 11.5. The eluate was collected in 0.5 ml aliquotsand neutralized with 1/20 volume of 1M sodium phosphate buffer, pH 6.8plus 0.05% Triton X-100.

A vpr-CNBr-Sepharose column was then constructed by coupling thepurified recombinant vpr to cyanogen bromide activated sepharose beads(Sigma). Recombinant vpr at 1 mg/ml was incubated with swelled beads for2 hours at 25 C in 10 mM NaHCO₃, 0.5M NaCl pH 8.3. The coupled beadswere blocked with 1M glycine. Cell lysates are obtained using TritonX-100 at a final concentration of 0.05% v/v with about 10⁷ cells of oneof the following types of cells: RD, U937, primary lymphocytes, primarymonocytes or macrophages. The vpr-CNBr-Sepharose column is loaded withthe lysates by letting the beads incubate with the lysates for at leastone hour. Elution was performed using first pre-elution buffer composedof 10 mM Sodium Phosphate buffer pH 6.8, followed by elution bufferconsisting of 100 mM glycine, pH 2.5. Elution fractions are neutralizedwith 1/20 volume of 1M sodium phosphate buffer, pH 8.0.

The eluate is isolated vpr. receptor protein which may be. visualizedusing silver stained SDS-PAGE where the vpr receptor runs at about 41kD. The isolated vpr receptor protein binds to vpr as well as humanglucocorticoid receptor (hGR) protein.

We claim:
 1. Essentially pure viral protein R receptor proteincharacterized by a molecular weight of about 41 kD as determined using12% SDS-PAGE, an ability to bind to viral protein R and solubility inTriton, or a fragment of said viral protein R receptor protein whichbinds to viral protein R.
 2. The protein of claim 1 wherein said proteinis characterized by a molecular weight of about 41 kD as determinedusing 12% SDS-PAGE, an ability to bind to viral protein R and solubilityin Triton.
 3. The protein of claim 1 wherein said protein is a fragmentof the protein which has a molecular weight of about 41 kD as determinedusing 12% SDS-PAGE, said fragment having the ability to bind to viralprotein R and solubility in Triton.
 4. A method of identifying compoundswhich inhibit binding of viral protein R to the viral protein receptorprotein of claim 1 which comprises the steps of:a) contacting in thepresence of a test compound, viral protein R protein or a fragmentthereof and said viral protein R receptor protein or a fragment thereof,wherein in the absence of said test compound said viral protein Rprotein or said fragment thereof binds to said viral protein R receptorprotein or said fragment thereof; b) determining the level of bindingand c) comparing that level to the level of binding that occurs whenviral protein R protein and said viral protein R receptor protein arecontacted in the absence of a test compound, wherein a decrease inbinding levels in the presence of said test compound indicates that thetest compound is a compound which inhibits binding of viral protein Rprotein to the viral protein R receptor protein of claim
 1. 5. A kit foridentifying compounds which inhibit binding of viral protein R proteinto the viral protein R receptor protein of claim 1 which comprisesa) afirst container which contains viral protein R protein or a fragmentthereof which binds viral protein R receptor protein or a fragmentthereof, and b) a second container which contains said viral protein Rreceptor protein or a fragment thereof which binds viral protein Rprotein or a fragment thereof.